DESCRIPTION (Applicant's abstract): The long-term goals of this proposal are to examine the structure, function, and mechanism of action for a set of enzymes required for the biosynthesis and catabolism of mammalian N-linked glycans. We have focused on the cx-mannosidases in the N-glycan pathway since they act as committed steps in the synthesis of complex oligosaccharides by determining the extent of mannose trimming, and in some cases appear to control the rate of degradation of unfolded glycoproteins in the endoplasmic reticUlum (ER). The processing and catabolic a-mannosidases can be organized into two multigene families, termed Class I and Class 2 mannosidases, that differ in primary sequence and biochemical characteristics. This application is focused exclusively on the Class 1 mannosidases. Three subfamilies of Class I mannosidases have emerged from our cloning studies. The ER mannosidase I subfamily cleaves a single residue from Man9GlcNAc2 to generate a specific Man8GlcNAc2 isomer. The Golgi mannosidase I subfamily cleaves Man9-8GlcNAc2 structures to Man5GlcNAc2. The HTM subfamily members, designated based on their "homology to mannosidases," do not appear to have an intrinsic hydrolase activity, but may have a lectin activity involved in glycoprotein degradation. Recently, both ER Man I and HTM proteins have been implicated as key players in the targeting of unfolded glycoproteins for disposal in the ER. This application will examine the mechanism used by the Class 1 mannosidases to recognize unfolded glycoproteins for disposal with a goal of understanding how intervention in the process can lead to enhanced protein stability in human genetic diseases characterized by the rapid degradation of unfolded glycoproteins. Four specific aims are addressed in this proposal. The first aim will characterize the novel mechanism of hydrolase action and specificity of the ER and Golgi mannosidases by a combination of mutagenesis, kinetic analysis, binding studies, and structural analysis. The second aim will assess the roles of specific domains and essential residues of HTM proteins using a yeast model system. The third aim will determine the substrate/ligand specificity and binding partners of the novel HTM proteins involved in ER glycoprotein degradation. The fourth aim will determine the effects of altering the expression levels of ER Man I and HTM proteins on the rates of degradation of model unfolded proteins in mammalian cells.